Lactoperoxidase is an oxidoreductase contained in mammalian milks such as cows milk, and other secretions such as saliva, lacrymal fluid, and respiratory tract mucus (for example, American Journal of Respiratory and Critical Care Medicine, U.S.A. Vol. 166, 2002, p.S57 to S61). Lactoperoxidase is a protein having a molecular weight of about 80,000. Lactoperoxidase has heme as a coenzyme per one molecule. Since the maximum absorption wavelength of this heme is about 412 nm, highly-purified lactoperoxidase exhibits a brown color (for example, British Journal of Nutrition, England, Vol. 84, 2000, p.S19 to S25).
It is reported that lactoperoxidase has various biological functions such as antibacterial properties, antiviral activity, antioxidative activity, anticancer activity, and immunoregulatory activity (for example, said British Journal of Nutrition, England, Vol. 84, 2000, p.S19 to S25, and Life Sciences, England, Vol. 43, 1988, p.739 to 745), and it is revealed that this is a very important protein in relation to host defense. Regarding the industrial application of such lactoperoxidase, there are disclosed techniques such as: use of lactoperoxidase, peroxide donor, and thiocyanate for the manufacture of a medicament for treating helicobacter pylori infection (for example, Published Japanese translation No. 2000-509367 of PCT); a preventive and therapeutic agent for infectious disease with pathogenic germs added to formula feed for cultured aquatic animals (for example, Japanese Patent No. 3103615); an aging preventing agent (for example, Japanese Patent No. 3103167); a hepatic function ameliorative agent (for example, Japanese Unexamined Patent Application, First Publication No. 2001-226289), prophylactic and therapeutic applications of peroxidases (for example, Published Japanese translation No. H06-501453 of PCT); and a therapeutic agent for corneal disorder (for example, Japanese Patent No. 2840795). Furthermore, there are disclosed techniques by the present inventors such as for: a urease-inactivating composition and a beverage (for example, Japanese Unexamined Patent Application, First Publication No. 2002-238554), and an intestinal flora improving agent and food and drink (for example, Japanese Unexamined Patent Application, First Publication No. 2003-246753).
Purification methods for lactoperoxidase in laboratory scale are reported in: Acta Chemica Scandinavica, Denmark, Vol. 23, 1969, p. 171 to 184; FEBS Letters, Holland, Vol. 110, 1980, p. 200 to 204; and Journal of Chromatography, Holland, Vol. 795, 1998, p. 277 to 287.
As a typical method thereof, there is known a method for: adding an acid such as hydrochloric acid into milk, to isoelectrically precipitate casein, so as to prepare whey serving as a supernatant; bringing the obtained whey into contact with a cation exchanger, so as to adsorb positively charged lactoperoxidase in the whey, into the cation exchanger; and next, washing the cation exchanger with a low salt buffer solution, and then desorbing the lactoperoxidase with a high salt buffer solution.
In a purification method in laboratory scale, in order to improve the lactoperoxidase purity, there is generally used a method for using a column which is highly densely filled with a cation exchanger in a gel form having small-diameter particles, and high-speed passing through is performed with a high pressure pump (for example, Journal of Chromatography, Holland, Vol. 795, 1998, p. 277 to 287).
On the other hand, if a column is filled with a cation exchanger having relatively large-diameter particles, and passing through is performed by means of natural drop without a high pressure pump, it takes more time (for example, Acta Chemica Scandinavica, Denmark, Vol. 23, 1969, p. 171 to 184, and FEBS Letters, Holland, Vol. 110, 1980, p. 200 to 204).
Together with the recent progress in isolation techniques at an industrial scale, it becomes possible to isolate and purify a high purity bioactive substance contained in milk, for mass production. In most cases, it is realistically difficult to scale up a protein purification method optimized at a laboratory scale into an industrial scale as it is. One of the main causes is that the property of an ion exchanger or a column generally used in a laboratory is not necessarily suitable for mass treatment of a raw material.
Furthermore, since addition of additives into milk materials tends to change the milk flavor and physical properties, it is not preferable to use additives for purifying a protein from milk materials. Furthermore, if a large amount of additives are used in order to wash a cation exchanger and/or to desorb a protein from the cation exchanger, it becomes necessary to remove these additives from the purified protein, and the production process becomes complicated.
As a production process for solving these problems in the production of a high purity protein from milk materials, there is already proposed by the present applicant, a production process for high purity bovine lactoferrin (for example, Japanese Examined Patent Application, Second Publication No. H06-13560).
Regarding the industrial production process for lactoperoxidase, the following are disclosed.
In the specification of U.S. Pat. No. 4,667,018, there is disclosed a process for purifying proteins such as lactoferrin and lactoperoxidase from milk or its milk derivative. In this process, there is disclosed a method for: bringing milk or its milk derivative into contact with a cation exchanger comprising cationic polysaccharides; washing the cation exchanger with a low salt solution; and then desorbing the proteins from the cation exchanger with a high salt solution. However, since the proteins produced in the method described in this Patent Document are obtained as a mixture, the purity is not high, and there is a problem in that high purity lactoperoxidase can not be produced.
In Japanese Patent No. 2985158, there is disclosed a method for recovering lactenin fraction having high activity. In this method described in this Patent Document, since lactoperoxidase is obtained as one component constituting lactenin, and contained in a protein mixture, there is also a problem in that high purity lactoperoxidase can not be produced.
In the specification of European Patent No. 0518448, as a method for isolating proteins from milk, there is disclosed a method of using a metal chelate carrier. In this method, since the isolated protein is a mixture comprising immunoglobulin, lactoferrin, and lactoperoxidase, there is a problem in that it is impossible to produce high purity lactoperoxidase.
In Japanese Patent No. 3403066, there is disclosed a method for recovering a cell-proliferating factor or a composition containing one or more kinds of cell-proliferating factors from milk or a milk derivative. However, since the composition obtained in this method is a mixture, there is also a problem in that this is not a process for producing high purity lactoperoxidase.
In Japanese Patent No. 2710283, as a method for selectively extracting a metal protein from whey, there is disclosed a method comprising a step for bringing whey into contact with inorganic porous particles (silica particles) coated with dextran comprising carboxyl groups or sulfonic groups. The purity of lactoperoxidase produced in this method is about 50% at the highest, and there is a problem in that, in order to produce high purity lactoperoxidase, it is necessary to increase the purity by another step.
In Japanese Patent No. 2553180, there is disclosed a process for extracting pure fractions of lactoperoxidase and lactoferrin from whey. In this process, as a means for solving the problem of clogging caused by volume change of a cation exchanger, a microfiltered whey is used as a milk material. In this process, since no milk material other than whey can be used, there is a problem of narrow application range. Furthermore, microfiltration is also required as a pretreatment of milk materials, complicating the production step. Moreover, a strong cation exchanger is used for the cation exchanger, and lactoperoxidase and lactoferrin are selectively desorbed by buffer solutions having different salt concentrations. This process requires, in order to wash the cation exchanger and to selectively desorb proteins, that the pH of the buffer solutions need to be adjusted, and therefore, a large amount of additives is used to prepare such buffer solutions. Moreover, there are various problems such that it is necessary to remove the additives from the purified proteins, serving as a factor of further complicating the step.
In Japanese Patent No. 2686831, there is disclosed a method for separating and purifying an iron binding protein using a strongly cationic sulfone group-introduced polysaccharides affinity carrier as a cation exchanger. In this method, a relatively highly purified (purity 85%) lactoperoxidase is produced. However, in a step for washing the cation exchanger after adsorption treatment, a washing treatment with a buffer solution adjusted to pH 5 or less is essential. Moreover, buffer solutions having respectively different salt concentrations of pH 5 or less are required for selectively desorbing lactoperoxidase and lactoferrin.
In Japanese Unexamined Patent Application, First Publication No. H05-202098, as a process for producing a bioactive substance from milk materials, there is disclosed a technique which can use either of a strong cation exchanger having sulfone groups and a weak cation exchanger having carboxyl groups. However, in this method, there is still a problem in that a buffer solution of pH 5 or less is required for enabling to wash the cation exchanger after adsorption treatment and to selectively desorb lactoperoxidase.
In U.S. Pat. No. 5,596,082, there is disclosed a process for isolating lactoferrin and lactoperoxidase from milk and milk products. In this patent, in order to enable to pass through milk and milk products at a high flow rate, a physically stable gel having large-diameter particles, is used as a cation exchanger. In this process, there is also a problem in that pH adjustment using a buffer solution is required for washing the cation exchanger after adsorption treatment and selectively desorbing lactoperoxidase. Since there is no description of the purity of lactoperoxidase produced in this patent, it is uncertain whether or not highly purified lactoperoxidase can be produced.
As described above, in most conventional processes for producing lactoperoxidase, lactoperoxidase recovered from milk materials is obtained as a mixture with other proteins, and the purity is not said to be sufficiently high. Moreover, in order to increase the purity of lactoperoxidase, other purification steps are required, thus requiring time and production costs therefor.
Moreover, even in the production process in which the purity of lactoperoxidase becomes relatively higher, there is also a problem in that pH adjustment is required in respective steps for; selectively adsorbing lactoperoxidase contained in milk materials, washing the cation exchanger, and selectively desorbing proteins, thus requiring use of a large amount of additives.